Fig. 4: Multiplexed photoconversion of green-to-red and NIR-to-far-red PCFPs for organelle tracking.

Assessment of the (a) spectral, (b) fluorescence lifetime compatibility of miRFP720 (gray-to-magenta color) and mEos2 (blue-to-orange color) when expressed in Hela cells. The fast FLIM lifetimes are representative of cells expressing mEos3.2-clathrin and LAMP1-miRFP720. c FLIM image reporting the crosstalk between LAMP1-miRFP720 and mEos2-Peroxisomes in the 620–670 nm detection band. The fluorescence lifetime information encoded in the colormap allows to distinguish the two proteins and, therefore, correctly segment the two labeled organelles. d Sequential scheme of recording, for the multicolor photoconversion imaging. The excitation and detection band are color coded to reflect the signal of the protein that is predominant under that excitation and detection condition. e Reactivity to the 405 nm light as photoconversion wavelength measured in Hela cells expressing LAMP1-miRFP720 (filled circles) and mEos3.2-clathrin (empty circles). The reactivity to 405 nm illumination is measured as the ratio between the fluorescence of the photoconverted state and the ground state. The graphs are normalized for comparison between the two PCFPs. The asterisk identifies the 405 nm power used to photoconvert both proteins simultaneously. Each data point is the mean and SD of the photoconversion yield for N = 25–50 organelles. f illustration of coupled photoconversion multiplexing scheme with a common PC wavelength. Through illumination with 405 nm light, green-to-red and NIR-to-far-red PCFPs can be combined to achieve spatially colocalized photoconversion at the subcellular level. g Coupled photoconversion experiment in HeLa cells, with the labeling of peroxisomes, mEos2-Peroxisomes, and lysosomes, LAMP1-miRFP720. Before photoconversion, all the fluorescence was emitted by the green (500–550 nm, cyan) and NIR (710–750 nm, gray) forms. h Upon illumination with 405 nm light (3.16 J/cm²), a confined region (dotted rectangle in g) of the cells was photolabeled, where the two proteins photoconverted to the red (570–590 nm, orange) and far-red (620–660 nm, magenta) forms, respectively. i A closer look at the photoconverted region and motility of the organelles disentangled over time for the photoconverted area (time interval 1 min). Scale bars, 5 µm. Representative images of 5 experiments. Source data are provided as a Source Data file.